SUPEROXIDE ANION GENERATION BY INSITU PERFUSED-RAT-LIVER - EFFECT OF INVIVO ENDOTOXIN

A simple method is described to monitor the superoxide dismutase (SOD)-inhibitable production of superoxide anion (O-2-.) in the liver. The isolated rat liver was perfused in situ with ferricytochrome c, and the reduction of this substrate during perfusion was determined. Within 30 s after the introduction of the substrate, significant reduction of ferricytochrome c was observed and stabilized at 2-4 min. A marked reduction of the substrate was observed in the livers of rats that received Escherichia coli lipopolysaccharide (LPS, 1 mg/kg) in vivo 3 h before liver perfusion. Ferricytochrome c reduction was inhibited by SOD, but not significantly with allopurinol or deferoxamine mesylate in the livers of LPS-treated rats. Control livers exhibited only a small reduction of the substrate, and this was not significantly inhibited by SOD. After in vivo LPS administration, 0-2-. production peaked in the liver at 3 h (6.6 nmol/min) and returned toward normal at 6 h (1 nmol/min) after endotoxin. The amount of O-2-. generated by the endotoxic livers was dose related. At 3 h post-LPS, neutrophil infiltration and necrotic areas were found in the histological sections of the liver with concomitant elevation of serum aminotransferases, indicating hepatic abnormalities during the early stage of endotoxemia. Phorbol myristate acetate in the perfusion system markedly enhanced 0-2-. generation in the endotoxic liver. These results show that the perfused rat liver can be used to measure O-2-. generation following in vivo stimuli. The data also demonstrate that O-2-. release after LPS treatment in vivo is a short and early event and may have an important role in hepatic injury in endotoxemic conditions.